Insulin binding to the insulin receptor (IR) initiates a signaling cascade that plays an essential role in glucose homeostasis. Disruptions of this metabolic pathway may result in diabetes, a disease that afflicted 8.4% of the U.S. population in 2011. A key step toward combating diabetes is to understand ligand-dependent IR signaling and to develop new pharmacologic agents that modulate the IR. Remarkably, despite extensive efforts spanning several decades, the molecular mechanisms of IR activation by the binding of insulin remain unelucidated.
IR, a member of the receptor tyrosine kinase superfamily, is a glycoprotein consisting of two α and two β subunits (α2β2) covalently linked by disulfide bonds. See, e.g., Siddle et al., Biochem Soc Trans (2001) 29:513-525. The extracellular domain (also called the ectodomain) of the IR comprises two a subunits and the N-terminal segment of the two β subunits, whereas the transmembrane domains and cytoplasmic tyrosine kinase (TK) domains comprise the C-terminal segments of the β subunits. The insulin-binding determinants reside entirely within the ectodomain, which consists of leucine-rich repeat domains L1 and L2 of the α chain, the intervening cysteine-rich (CR) domain, and three fibronectin type III domains, namely Fn0, Fn1, and Fn2.
Although insulin itself was the first peptide hormone to be structurally elucidated by X-ray crystallography, see, e.g., Blundell et al., Nature (1971) 231:506-511, and has been the subject of extensive structural investigations over the past 50 years, the molecular mechanism of IR activation by insulin remains unelucidated. Menting et. al. elucidated a structure of insulin bound to the L1 domain and the aCT peptide. See Nature, 2013, 493: 241-248. Insulin binding to IR is characterized by exceptionally high-affinity binding (pM range) and negative cooperativity. See, e.g., De Meyts et al., Biochem Biophys Res Commun (1973) 55:154-161; De Meyts et al., Diabetologia (1994) 37 Suppl 2:S135-148. Evidence suggests that there are two insulin binding sites on the IR, site 1 and 2, wherein each site 1 on one monomer of IR is close to site 2′ on the second monomer, and binding of insulin to site 1 induces its subsequent binding to site 2′, which causes a conformational change of the IR ectodomain, leading to a reduction of the distance between the two intercellular TK domains, thereby facilitating autophosphorylation. Several lines of evidence have shown that site 1 on the IR is formed by the central β-sheet of the L1 domain and a C terminal α-subunit peptide segment termed αCT (aa704-aa719), while site 2 is believed to reside at the loop region between Fn0 and Fn1, since it faces site 1 of the other monomer in the dimeric structure of the IR ectodomain. See, e.g., Huang et al., J Mol Biol (2004) 341:529-551; Mynarcik et al., J Biol Chem (1997) 272:18650-18651; Kurose et al., J Biol Chem (1994) 269:29190-29197; Mynarcik et al., J Biol Chem (1996) 271:2439-2442.
Peptides that bind site 1 are either agonists or antagonists, while peptides that bind site 2 are antagonists. Further optimization of site 1 and site 2 peptides by dimerization has identified either potent agonists or antagonists (pM IR binding affinity) depending on the mode of linkage. See, e.g., Schaffer et al., Proc Natl Acad Sci USA (2003) 100:4435-4439; Schaffer et al., Biochem Biophys Res Commun (2008) 376:380-383; Jensen et al., Biochem J (2008) 412:435-445. Intriguingly, though these peptides show no sequence similarity with insulin, a close relationship was proposed between the site 1 peptide and α-CT, indicating site 1 peptides are α-helical. See, e.g., Smith et al., Proc Natl Acad Sci USA (2010) 107:6771-6776; Menting et al., Biochemistry (2009) 48:5492-5500. Although these peptides are attractive candidates for insulin mimetics, the potential for therapeutic use is limited due to their inherent structural instability; therefore, there remains a need for stabilized peptides that bind the IR for therapeutic as well as scientific purposes.